BK ZERO isoform HEK293 stably transfected cell lines differing 3’UTRs to assess miR-9 regulation

Research has identified the large conductance voltage- and calcium-activated potassium channel (BK) as a key regulator of neuronal excitability genetically associated to behavioral alcohol tolerance. Sensitivity to ethanol at the molecular level is characterized by acute potentiation of channel activity. BK isoforms show variations in alcohol sensitivity and are differentially distributed on the plasma membrane surface in response to prolonged exposure. MicroRNA (MiRNA) targeting of alcohol-sensitive isoforms coupled with active internalization of BK channels in response to ethanol are believed to be key in establishing homeostatic adaptations that produce persistent changes within the plasma membrane of neurons. In fact, microRNA 9 (miR-9) upregulated expression is a key event in persistent alcohol tolerance mediating acute EtOH desensitization of BK channels. The exact nature of these interactions remains a current topic of discussion. To further study the effects of miR-9 on the expression and distribution of BK channel isoforms we designed an experimental model by transfecting human BK channel isoforms ZERO heterologous constructs in human embryonic kidney cells 293 (HEK293) cells respectively expressing 2.1 (miR-9 responsive), 2.2 (unresponsive) and control (no sequence) 3’untranslated region (3’UTR) miRNA recognition sites. We used imaging techniques to characterize the stably transfected monoclonal cell lines, and electrophysiology to validate channel activity. Finally, we used immunocytochemistry to validate isoform responsiveness to miR-9. Our findings suggest the cell lines were successfully transfected to express either the 2.1 or 2.2 version of ZERO. Patch clamp recordings confirm that these channels retain their functionality and immunohistochemistry shows differential responses to miR-9, making these cells viable for use in future alcohol dependence studies.

Lipofectamine 2000 (11668027 ThermoFisher, Lithuania) Blue Heron's plasmid cDNAs: Protocol: Genemaker® Gene Synthesis Platform from Blue Heron, LLC The company uses Genemaker® which is a fully automated gene synthesis platform designed to facilitate the entire gene synthesis process.The process began with inputting the desired gene sequence into the website ordering page.The company proceeded to optimize the gene build based on the entered sequence using a proprietary algorithm.

SAFETY WARNINGS
Please see SDS (Safety Data Sheet) for hazards and safety warnings. 1 Thaw cells and then plate them as follows: 1.1 Resuspend in 1mL of modified DMEM media composed of Dulbecco's modified Eagle's medium (D5796, Milwaukee, WI, USA) with 10% fetal bovine serum (FBS), 2.52mM HEPES; 10 units and 10µg/mL of Penicillin/Streptomycin (Pen/Strep), and 1mM Na-pyruvate.

1.3
Flood with DMEM up to a final volume of 2mL.

2
Place cells in a Sanyo CO 2 incubator (37.0 o C/5% CO 2 level) and allow them to reach full confluency replacing the media every 3-5 days.

3
Once the cells reach 70-90% confluency suction media from the plates and add Trypsin-EDTA 1:10 diluted in 1X PBS to solubilize the cells.Apply gentle shaking to the plates to allow for the separation of the cells from the plate.Add a volume of DMEM to the plate equal to the added volume of trypsin to prevent further cell lysis.

5
Remove 10mL tubes from the chemical hood and centrifuge at 1,380g for 60 seconds.

6
Place the tubes inside the chemical hood and aspirate the supernatant.

8
Extract 10µL of each tube and mix with equal volume of Trypan Blue Dye.

9
Add a single droplet of the Trypan Blue mixture to a counting slide and place in an automated cell counter.

10
While the cells are being counted, add an additional 9mL of modified DMEM to the tube and gently resuspend, for a total volume of 10mL.

11
Once counted add the appropriate seeding density (0.3x10 6 cells) to the 35mm petri dishes and place them in the incubator and allow them to reach 70-90% confluence.
12 Reseed cells onto a 24-well plate and incubate until they reach 70-90% confluence. 13 Once cells reach 70-90% confluence remove the plates from the incubator and place them in the cell culture hood.

14
Wash the plates 3 times with 1X PBS for 30 seconds.Gently swirl between washes.

15
Add serum-deprived DMEM (no FBS) to the plates and place them in the incubator for 24 hours. 16 After incubation prepare a 100µL solution of Opti-MEM reduced serum media containing 0.8µg of cDNA BK ZERO isoform vector and 2.0µL of Lipofectamine 2000 (11668027, Thermofisher, NY, USA).17 Incubate mixture for 5 minutes at room temperature before use.

18
Add the mixture to each well in the 24-well plate and place them in the incubator for 16 hours to allow for transfection to occur.

19
After the incubation period, completely exchange transfection media with DMEM.

20
Wash 3 additional times with 1X PBS and place in modified DMEM for 24 hours in the incubator.

22
Once cells reach 70-90% confluence remove the dishes from the incubator and place them in the cell culture hood.

23
Wash the plates 3 times with 1X PBS for 30 seconds.Gently swirl between washes.

24
Add serum-deprived DMEM (no FBS) to the plates and place them in the incubator for 24 hours.

25
After incubation prepare a 100µL solution of Opti-MEM reduced serum media containing 0.8µg of cDNA BK ZERO isoform vector and 2.0µL of Lipofectamine 2000 (11668027, Thermofisher, NY, USA).

26
Incubate mixture for 5 minutes at room temperature before use.

27
Add the mixture to each well of the 24-well plate and place them in the incubator for 24 hours to allow for transfection to occur.

28
After the incubation period, completely replace the transfection media with DMEM the reduced.

29
Wash 3 additional times with 1X PBS and place in modified DMEM for 24 hours in the incubator.
dx.doi.org/10.17504/protocols.io.yxmvm3ek5l3p/v2Oct 29 2023 8 The optimization aimed to balance complexity reduction and protein expression enhancement.Once the Oligonucleotides are synthesized the are amplified via PCR synthesis techniques, utilizing both solution and solid substrate technologies.The synthesized genes are then cloned into the vector of choice.The cloned gene is sequence verified to ensure 100% accuracy and ship to us.

Table .
Summary of Transfected Plasmids.